Pyropheophorbides and their use in photodynamic therapy

ABSTRACT

Pyropheophorbide compounds are injected into a host and accumulate in tumor tissue to a higher degree than surrounding normal tissues. When the pyropheophorbide compounds are exposed to a particular wavelength of light the compounds become cytotoxic and destroy the tumor or diseased tissue without causing irreversible normal tissue damage. The pyropheophorbide compounds have shown improved results as compared to drugs currently used in photodynamic therapy. Further, they absorb light further in the red, optimizing tissue penetration and are retained in the skin for short time periods relative to other drugs used in photodynamic therapy.

CROSS-REFERENCES

This application is a continuation of our earlier filed application Ser.No. 07/973,174 filed Nov. 9, 1992, now issued as U.S. Pat. No. No.5,314,905; which is a divisional of Ser. No. 07/822,409 filed Jan. 17,1992, now issued as U.S. Pat. No. 5,198,460; which is acontinuation-in-part of Ser. No. 07/597,786 filed Oct. 15, 1990, nowissued as U.S. Pat. No. 5,093,349; which is a continuation of Ser. No.07/221,804 filed Jul. 20, 1988, now issued as U.S. Pat. No. 5,002,962;all of which are incorporated herein by reference and to which we claimpriority under 35 U.S.C. §120.

FIELD OF THE INVENTION

This invention relates generally to photosensitive therapeutic compoundsand photodynamic therapy (PDT). More particularly, the invention relatesto pyropheophorbides, formulations that contain such and their use inthe treatment of cancer.

BACKGROUND OF THE INVENTION

As described in U.S. Pat. No. 5,002,962, porphyrin related compoundsaccumulate at higher concentrations in tumor tissue as compared tonormal tissue, and that irradiation of these compounds using light ofthe proper wavelength results in an energized form which, upon decay,results in cytotoxicity. It is believed that excitation of the porphyrinor related material results in the formation of singlet oxygen which isin fact the toxic agent. However, the compounds administered apparentlydo not degrade in this process.

Literature relating to the use of "hematoporphyrin derivative" (HPD)describes this process utilizing a preparation obtained whenhematoporphyrin dichloride is treated using the procedure of Lipson, R.L., et al, J National Cancer Inst (1961) 26:1-8. More recently, it hasbeen shown that if this hematoporphyrin derivative is treated at asuitable pH, aggregation occurs and the active material in the mixturecan be prepared in crude form as a size segregated aggregate (see, forexample, U.S. Pat. No. 4,649,151, incorporated herein by reference).This preparation is commercially available under the trademarkPhotofrin.

The preparation marketed as the Photofrin composition is a mixture. Themixture contains porphyrins joined by ether linkages (Dougherty, T. J.,et al, Adv Exp Med Bio (1983) 160:3-13), and more recently, Kessel, D.,et al Photochem Photobiol (1987) 46:463-568, has shown that ester linkedporphyrins are contained in this mixture as well. Scourides, P. A., etal, Cancer Res (1987) 47:3439-3445 have synthesized an oligomericmixture of ether linked porphyrins starting from hematoporphyrindimethyl esters. The mixture was active in PDT, but was as complex amixture as the Photofrin preparation. Dimers of hematoporphyrin joinedby ester linkages have also been prepared by Pandey, R. K., et al,Cancer Res (in press) and the dimers prepared were shown to be absentfrom the mixture in the Photofrin composition as well as inactive in anin vitro assay.

Thus, it is known in the art that some elements of a mixture preparedwhen HPD is aggregated and segregated into higher molecular weightcomponents are active in photodynamic therapy. Earlier, the presentinventors prepared single compound compositions useful in PDT asdisclosed in U.S. Pat. No. 5,002,962. The purified and definedcompositions disclosed in U.S. Pat. No. 5,002,962 are useful inphotodynamic therapy as are compounds and methods disclosed in U.S. Pat.Nos. 4,920,143 and 4,883,790.

SUMMARY OF THE INVENTION

Pyropheophorbide compounds and pharmaceutical compositions containingsuch compounds can be used in methods of photodynamic therapy. Thepyropheophorbides are encompassed by the following general structuralformula I or II. ##STR1## wherein R₁ is CH₂ OR₂ where R₂ is a primary orsecondary alkyl containing 1 to 20 carbons; and R₃ is --CH₂ CH₂ CO₂ R₄where R₄ is H or an alkyl containing 1 to 20 carbons. Other compounds ofthe invention are encompassed by formula II as follows: ##STR2## whereinR₅ is --OR₅ where R₆ is a primary or secondary alkyl containing 1 to 20carbons and R₇ is --CH₂ CH₂ CO₂ R₈ where R₈ is H or an alkyl containing1 to 20 carbons. Particularly preferred compounds are where R₅ is--O-hexyl and R₇ is --CH₂ CH₂ CO₂ H or --CH₂ CH₂ CO₂ CH₃. Thepyropheophorbides of the invention are combined with excipients toprovide pharmaceutically acceptable formulations suitable for use inphotodynamic therapy.

The invention also includes methods of synthesizing compounds of formulaI and II.

The invention includes injectable pharmaceutical compositions containingthe pyropheophorbide compounds of the invention as active ingredientsand to methods of conducting photodynamic therapy using the compoundsand compositions of the invention.

The invention also includes the pyropheophorbide compounds of theinvention conjugated to a ligand which is capable of binding a specificreceptor such as a cellular receptor, or an antibody which is capable ofbinding to a particular antigen and to compositions containing theseconjugates and methods of conducting photodynamic therapy using theconjugates and their compositions.

A primary object of the invention is to provide pyropheophorbidecompounds, pharmaceutical compositions containing such compounds andmethods of treatment carried out using such compounds in a photodynamictherapy.

Other objects are to provide methods of treating humans with tumor cellswhich cells replicate abnormally fast, treating atherosclerosis orinactivating bacteria or virus infections.

A feature of the present invention is that the pyropheophorbidecompounds of the invention absorb light further into the red portion ofthe spectrum as compared with conventional compounds used inphotodynamic therapy.

An advantage of the present invention is that the pyropheophorbidecompounds and pharmaceutical compositions of the invention optimizetissue penetration and are retained in the skin for relatively shortperiods of time as compared with other compounds used in photodynamictherapy.

Another advantage of the present invention is that the pyropheophorbidecompounds of the invention have a greater toxicity with respect to tumorcells and diseased tissue as compared with the toxicity of conventionalcompounds used in photodynamic therapy.

Another advantage of the invention is that the pyropheophorbides can besynthesized as free acids (e.g. in formula I or II when R₃ or R₇ is--CH₂ CH₂ CO₂ H) allowing ease in formulation without the need forliposomes or detergents.

Another advantage of the invention is the pyropheophorbide of theinvention are active at very low doses of injected material as comparedto conventional photosensitizers used in photodynamic therapy.

These and other objects, advantages and features of the presentinvention will become apparent to those persons skilled in the art uponreading the details of the structure, synthesis and usage as more fullyset forth below, reference being made to the accompanying structuralformals forming a part herein wherein like symbols refer to likemolecular moieties throughout.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a FAB mass spectrum of the compound of formula II(a).

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS OF THE INVENTION

Before the present pyropheophorbide compounds, pharmaceuticalcompositions, methods of synthesizing and using such compounds aredisclosed, it is to be understood that this invention is not limited tothe particular compounds, compositions, methods of use or synthesis asdescribed as such may, of course, vary. It is also to be understood thatthe terminology used herein is for the purpose of describing particularembodiments only, and is not intended to be limiting since the scope ofthe present invention will be limited only by the appended claims.

It is to be noted that as used in this specification and the appendedclaims, the singular forms "a", "and" and "the" include plural referenceunless the context clearly dictates otherwise thus, for example,reference to "a pyropheophorbide" includes mixtures of suchpyropheophorbides, reference to "an antibody" includes mixtures of suchantibodies and reference to "the method of treatment" includes referenceto like methods which will become apparent to those skilled in the artupon reading this disclosure.

Unless defined otherwise all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art of photodynamic therapy. Although any methods and materialssimilar or equivalent to those described herein may be used in thepractice or testing of the present invention, attempts have been made todescribe preferred methods and materials below.

The essence of the invention is the disclosure of novel compounds andpharmaceutical compositions containing such compounds which have beenfound to be highly effective in the treatment of cancer when used inconnection with a photodynamic therapy. More specifically, the compoundsare pyropheophorbide compounds which are encompassed by the followinggeneral structural formulae I and II. ##STR3## wherein R₁ is CH₂ OR₂where R₂ is a primary or secondary alkyl containing 1 to 20 (preferably5-20) carbons; and R₃ is --CH₂ CH₂ CO₂ R₄ where R₄ is H or an alkylcontaining 1 to 20 carbons. Preferred compounds are when R₁ is --CH₂--O-hexyl and R₃ is --CO₂ CH₃ or --CO₂ H. Other compounds of theinvention are encompassed by formula II as follows: ##STR4## wherein R₅is --OR₆ where R₆ is a primary or secondary alkyl containing 1 to 20(preferably 5-20) carbons and R₇ is --CH₂ CH₂ CO₂ R₈ where R₈ is H or analkyl containing 1 to 20 carbons. Particularly preferred compounds arewhere R₅ is --O-hexyl and R₇ is --CO₂ H or --CO₂ CH₃.

The pyropheophorbide compounds of structural formulae I and II can beformulated into pharmaceutical compositions and administered to patientsin therapeutically effective amounts in order to treat cancer.

Although the invention encompasses all of the compounds of structuralformulae I and II it has been found that the compound of structuralformula IIa is particularly effective in the treatment of cancer whenused in connection with photodynamic therapy. Structural formula IIa isput forth below: ##STR5##

A generalized reaction scheme for the synthesis of the compound ofstructural formula IIa is put forth below: ##STR6##

Starting Materials

The starting material for preparation of the red light-absorbingcompounds is methyl pheophorbide-a, which is isolated from Spirulinadestridratada by the method of Smith and Goff (D. Goff, Ph.D. Thesis,Univ. of Calif., Davis, Calif. 95616, 1984 incorporated herein byreference). Briefly, 500 gm dried Spirulina was slurried in a largevolume of acetone and then liquid nitrogen was added to form a frozenslush. The slush was transferred to a 3-necked, 5-liter round bottomflask and heated to reflux under nitrogen with stirring for 2 hours. Themixture was filtered through Whatman paper on a Buchner funnel withextensive acetone washing. The extraction and filtration process wasrepeated 2 more times; all green color could not be removed from thesolid.

The green filtrate was evaporated and purified by flash chromatographyon Grade V neutral Alumina, eluting first with n-hexane to remove a fastrunning yellow band and then with dichloromethane to obtain the majorblue/gray peak containing pheophytin-a. Treatment of pheophytin-a with500 ml sulfuric acid in methanol for 12 hours at room temperature in thedark under nitrogen was followed by dilution with dichloromethane. Thereaction mixture was rinsed with water and then 10% aqueous sodiumbicarbonate and the organic layer was dried, evaporated, and the residuerecrystallized from dichloromethane/methanol to obtain 1.8 gm methylpheophorbide-a. Methyl pheophorbide-a appears to be inactive in the invivo tumorcidal activity assay when injected at a dose of 5 mg/kg.

Conjugates and Labeled Pyropheophorbides

In addition to using compositions which consist essentially of theabove-defined compounds or preparations as active ingredient, it ispossible to use derivatized forms in order to provide a specifictargeting mechanism. Commonly used target-specific components includemonoclonal antibodies and ligands which bind to a cellular receptor. Thecompositions can also be conveniently labeled.

The target-specific component can then be, for example, animmunoglobulin or portion thereof or a ligand specific for a particularreceptor. The immunoglobulin component can be any of a variety ofmaterials. It may be derived from polyclonal or monoclonal antibodypreparations and may contain whole antibodies or immunologicallyreactive fragments of these antibodies such as F(ab')₂, FAB, or FAB'fragments. Use of such immunologically reactive fragments as substitutesfor whole antibodies is well known in the art. See, for example,Spiegelberg, H. L., in "Immunoassays in the Clinical Laboratory" (1978)3:1-23 incorporated herein by reference.

Polyclonal anti-sera are prepared in conventional ways by injecting asuitable mammal with antigen to which antibody is desired, assaying theantibody level in serum against the antigen, and preparing anti-serawhen the titers are high. Monoclonal antibody preparations may also beprepared conventionally such as by the method of Koehler and Milsteinusing peripheral blood lymphocytes or spleen cells from immunizedanimals and immortalizing these cells either by viral infection, byfusion with myelomas, or by other conventional procedures, and screeningfor production of the desired antibodies by isolated colonies. Formationof the fragments from either monoclonal or polyclonal preparations iseffected by conventional means as described by Spiegelberg, H. L.,supra.

Particularly useful antibodies include the monoclonal antibodypreparation CAMAL1 which can be prepared as described by Malcolm, A., etal, Ex Hematol (1984) ! 2:539-547; polyclonal or monoclonal preparationsof anti-M1 antibody as described by New, D., et al, J Immunol (1983)130:1473-1477 (supra) and B16G antibody which is prepared as describedby Maier, T., et al, J Immunol (1983) 131:1843; Steele, J. K., et al,Cell Imnunol (1984) 90:303 all of which publications are incorporatedherein by reference.

The foregoing list is exemplary and certainly not limiting; once thetarget tissue is known, antibody specific for this tissue may beprepared by conventional means. Therefore the invention is applicable toeffecting toxicity against any desired target.

The ligand specific for receptor refers to a moiety which binds areceptor at cell surfaces, and thus contains contours and chargepatterns which are complementary to those of the receptor. It is wellunderstood that a wide variety of cell types have specific receptorsdesigned to bind hormones, growth factors, or neurotransmitters.However, while these embodiments of ligands specific for receptor areknown and understood, the phrase "ligand specific for receptor," as usedherein, refers to any substance, natural or synthetic, which bindsspecifically to a receptor.

Examples of such ligands include the steroid hormones, such asprogesterone, estrogens, androgens, and the adrenal cortical hormones;growth factors, such as epidermal growth factor, nerve growth factor,fibroblast growth factor, and so forth; other protein hormones, such ashuman growth hormone, parathyroid hormone, and so forth; andneurotransmitters, such as acetylcholine, serotonin, and dopamine. Anyanalog of these substances which succeeds in binding to the receptor isalso included.

The conjugation of the target-cell-specific component to the compoundsof the invention can be effected by any convenient means. For proteins,such Ig and certain receptor ligands, a direct covalent bond betweenthese moieties may be effected, for example, using a dehydrating agentsuch as a carbodiimide. A particularly preferred method of covalentlybinding the compounds of the invention to the immunoglobulin moiety istreatment with 1-ethyl-3-(3-dimethylamino propyl) carbodiimide (EDCI) inthe presence of a reaction medium consisting essentially of dimethylsulfoxide (DMSO).

Of course, other dehydrating agents such as dicyclohexylcarbodiimide ordiethylcarbodiimide could also be used as well as conventional aqueousand partially aqueous media.

Nonprotein receptor ligands can be conjugated to be dimers and trimersaccording to their relevant functional groups by means known in the art.

The active moieties of the conjugate may also be conjugated throughlinker compounds which are bifunctional, and are capable of covalentlybinding each of the two active components. A large variety of theselinkers is commercially available, and a typical list would includethose found, for example, in the catalog of the Pierce Chemical Co.These linkers are either homo- or heterobifunctional moieties andinclude functionalities capable of forming disulfides, amides,hydrazones, and a wide variety of other linkages.

Other linkers include polymers such as polyamines, polyethers, polyaminealcohols, derivatized to the components by means of ketones, acids,aldehydes, isocyanates, or a variety of other groups.

The techniques employed in conjugating the active moieties of theconjugate-to the target-specific component include any standard meansand the method for conjugation does not form part of the invention.Therefore, any effective technique known in the art to produce suchconjugates falls within the scope of the invention, and the linkermoiety is accordingly broadly defined only as being either a covalentbond or any linker moiety available in the art or derivable therefromusing standard techniques.

The compounds of the invention per se or the conjugates may be furtherderivatized to a compound or ion which labels the drug. A wide varietyof labeling moieties can be used, including radioisotopes andfluorescent labels. Radioisotope labeling is preferred, as it can bereadily detected in vivo.

The compounds which are alone or are conjugates with a specific bindingsubstance can be labeled with radioisotopes by coordination of asuitable radioactive cation in the porphyrin system. Useful cationsinclude technetium and indium. In the conjugates, the specific bindingsubstances can also be linked to label.

Administration and Use

In general, the pyropheophorbide compounds of the invention areadministered to a host such as a human suffering from cancer intherapeutically effective amounts by any suitable means such asinjection which may be IV or IM or may be administered transdermally.The pyropheophorbide compounds of the invention accumulate in tumorcells to a much higher degree than they accumulate in surrounding normaltissues. After being provided with sufficient time so as to accumulatein the tumor tissue, the pyropheophorbide compounds are exposed to aparticular wavelength of light which causes the compounds to becomecytotoxic, thus destroying the tumor or diseased tissue which thepyropheophorbide compounds have accumulated in. This is accomplishedwithout causing irreversible damage to surrounding normal tissueswherein there has not been an accumulation of the pyropheophorbidecompounds.

The compounds and their conjugates with target-specific substances ofthe invention are useful, in general, in the manner known in the art forhematoporphyrin derivative and for Photofrin II compositions. Thesecompositions are useful in sensitizing neoplastic cells or otherabnormal tissue to destruction by irradiation using visible light--uponphotoactivation, the compositions have no direct effect, nor are theyentered into any biological event; however the energy of photoactivationis believed to be transferred to endogenous oxygen to convert it tosinglet oxygen. This singlet oxygen is thought to be responsible for thecytotoxic effect. In addition, the photoactivated forms of porphyrinfluorescence which fluoresce can aid in localizing the tumor. Thus, thedimer and trimer compounds of the invention are not consumed or alteredin exerting their biological effects.

Typical indications, known in the art, include destruction of tumortissue in solid tumors, dissolution of plaques in blood vessels (see,e.g., U.S. Pat. No. 4,512,762); treatment of topical conditions such asacne, athlete's foot, warts, papilloma, and psoriasis and treatment ofbiological products (such as blood for transfusion) for infectiousagents, since the presence of a membrane in such agents promotes theaccumulation of the drug. Other uses include treating humans sufferingfrom atherosclerosis and inactivating bacaterial or viral infections.

The compositions are formulated into pharmaceutical compositions foradministration to the subject or applied to an in vitro target usingtechniques known in the art generally. A summary of such pharmaceuticalcompositions may be found, for example, in Remington's PharmaceuticalSciences, Mack Publishing Co., Easton, Pennsylvania, latest edition. Thecompositions, labeled or unlabeled, can be administered systemically, inparticular by injection, or can be used topically.

Injection may be intravenous, subcutaneous, intramuscular, or evenintraperitoneal. Injectables can be prepared in conventional forms,either as liquid solutions or suspensions, solid form suitable forsolution or suspension in liquid prior to injection, or as emulsions.Suitable excipients are, for example, water, saline, dextrose, glyceroland the like. Of course, these compositions may also contain minoramounts of nontoxic auxiliary substances such as wetting or emulsifyingagents, pH buffering agents and so forth.

Systemic administration can also be implemented through implantation ofa slow release or sustained release system, by suppository, or, ifproperly formulated, orally. Formulations for these modes ofadministration are well known in the art, and a summary of such methodsmay be found, for example, in Remington's Pharmaceutical Sciences(supra).

If the treatment is to be localized, such as for the treatment ofsuperficial tumors or skin disorders, the compositions may be topicallyadministered using standard topical compositions involving lotions,suspensions, or pastes.

The quantity of compound to be administered depends on the choice ofactive ingredient, the condition to be treated, the mode ofadministration, the individual subject, and the judgment of thepractitioner. Depending on the specificity of the preparation, smalleror larger doses may be needed. For compositions which are highlyspecific to target tissue, such as those which comprise conjugates witha highly specific monoclonal immunoglobulin preparation or specificreceptor ligand, dosages in the range of 0.05-1 mg/kg are suggested. Forcompositions which are less specific to the target tissue, larger doses,up to 1-10 mg/kg may be needed. The foregoing ranges are merelysuggestive, as the number of variables in regard to an individualtreatment regime is large and considerable excursions from theserecommended values are expected. Further, because of slight solubilityin water, certain compounds of the invention may be administereddirectly in saline or 5% glucose solution, thus avoiding thecomplications of detergents or other solubilizing agents.

Those skilled in the art of photodynamic therapy and compounds relatedto the present invention will be better able to determine an appropriatedosage and overall dosage regime when taking a number of factors intoconsideration. For example, the size, weight and condition of thepatient must be considered as must be the responsiveness of the patientand their disease to the particular therapy. It is believed that evenrelatively small doses administered a single time can have a beneficialeffect. Further, extremely large doses could, of course, be toxic.Accordingly, rather than providing specific information on dosage amountand intervals between dosing, attention should be paid to conventionalfactors used in determining such dosing while considering that thepyropheophorbide compounds of the invention have a greater degree oftoxicity with respect to tumor cells and therefore can generally beadministered in smaller amounts than the conventional compounds used inconnection with photodynamic therapy.

EXAMPLES

The following examples are put forth so as to provide those of ordinaryskill in the art with a complete disclosure and description of how tomake the pyropheophorbide compounds and pharmaceutical compositions ofthe invention and are not intended to limit the scope of what theinventors regard as their invention. Efforts have been made to ensureaccuracy with respect to numbers used (e.g., amounts, temperature,etc.), but some experimental errors and deviations should be accountedfor. Unless indicated otherwise, parts are parts by weight, temperatureis in degrees Centigrade, and pressure is at or near atmospheric.

EXAMPLE 1

Methyl pyropheophorbide-a (2): Methyl pheophorbide-a (1, 1.0 g) wasobtained from alga Spirulina destridratada by following the proceduredescribed in K. M. Smith, D. A. Goff and D. J. Simpson, J. Am. Chem.Soc., 1985, 107, 4941-4954; and R. K. Pandey, D. A. Bellnier, K. M.Smith and T. J. Dougherty, Photochem. Photobiol., 1991, 53, 65-72, bothof which are incorporated herein by reference. The methyl pheophorbide-awas heated under reflux in collidine (100 ml) for 90 min during slowpassage of a stream of nitrogen. See G. W. Kenher, S. W. McCombie and K.M. Smith, J. Chem. Soc. Perkin Trans. 1973, 1, 2517-2523, incorporatedherein by reference. The solution is evaporated (0.1 mm Hg) and theresidue was recrystallized from dichloromethane/methanol. Yield 820 mg;91%, m.p. 217°-219° C., lit. 220°-225° C.; H. Fisher and A. Stern, DieChemie des pyrrole, vol II, Part 2, pp. 64 and 74, Akademische Verlag,Leipzig incorporated herein by reference. Vis: (max) 410 (112 000); 508(11 000); 536 (9 600); 610 (8 200); 666 (45 000); NMR, ppm; 9.50, 9.38,8.52 (each s, 3H, 3 meso H); 7.95-8.05 (m, 1H, CH═CH₂); 6.30 and 6.15(each s, 1H, CH--CH₂); 5.27 to 5.12 (q, 2H, 10-CH₂); 4.50 (m, 8-HO; 4.28(m, 7-H); 3.70 (q, 2H, CH₂ CH₃); 3.68 (s, 3H, CHCHCO₂ CH₃); 3.62, 3.40,3.22 (each s, 3H, 3CH₃); 2.70 (7a-H); 2.31(7a'-H); 2.56 (7b-H); 2.29(7b'-H); 1.82 (d, 3H, 8-CH₃); 1.70 (t, 3H, CH₂ CH₃); -1.70 (s, 2H,2 NH)

Pyropheophorbide-a (3): Methyl pyropheophorbide-a (2, 250 mg) wasdissolved in distilled tetrahydrofuran (50 ml) and 4N HCl (125 ml) wasadded in one lot. The reaction mixture was stirred under nitrogenatmosphere at room temperature for 4 hours. The reaction was monitoredby analytical tlc (silica plates), using 10% methanol/dichloromethane asa mobile phase. The reaction mixture was then poured in ice water,extracted with dichloromethane. The dichloromethane layer was washedseveral times with water (3×200 ml). The organic layer was separated anddried over anhydrous sodium sulfate. Evaporation of the solvent gave aresidue, which was crystallized from dichloromethane/hexane. Yield, 225mg. The purity of the compound was ascertained by tlc and the structurewas confirmed by NMR spectroscopy. The NMR data were similar asdescribed for 2 except the resonances for the --OCH₃ protons of thepropionic ester (--CH₂ CH₂ CO₂ CH₃) were missing.

Methyl-2-{1(O-hexyl)ethyl}-devinyl pyropheophorbide (4):Pyropheophorbide-a (2, 200 mg) was dissolved in 30% HBr/acetic acid (5.0ml) and the reaction mixture was stirred in a glass stoppered flask(rubber septum can also be used) at room temperature for 2.5 hours. Thesolvent was removed under high vacuum (1 mm Hg) and the resulting1-bromo ethyl derivative was immediately treated with n-hexanol (3.0 ml)under nitrogen atmosphere. The reaction mixture was stirred at roomtemperature for 45 min, diluted with dichloromethane (100 ml). Thedichloromethane layer was washed with water (3×200 ml) till the aqueousphase is neutral and then dried over anhydrous sodium sulfate.Evaporation of the solvent gave a residue, which was chromatographedover Alumina Grade III (6% water/neutral Alumina) and eluted withdichloromethane. The first fraction was a mixture of the startingmaterial and the desired product (minor quantity). Further elution withsame solvent gave the desired product. The appropriate eluates werecombined. Evaporation of the solvent afforded a sticky solid, which canbe crystallized from dichloromethane/hexane. Yield 70%. (see Scheme-1),Vis,(max); 408 (90 000); 471 (3 200), 506 (8600); 536 (8,500); 604(7,250); 660 (41 500). NMR, ppm; 9.79, 9.51, 8.53 (each s, 1H, meso H);5.90 (q, 2H, --CH (O-hexyl)CH₃ ; 5.08-5.30 (q, 2H, 10-CH₂); 4.47 (m,8H); 4.29 (m, 7-HO; 3.75 (q, 2H, CH₂ CH₃); 3.67 (s, 3H, CH₂ CH₂ CO₂CH₃), 3.67 (s, 6H, 2×CH₃); 3.38 and 3.27 (each s, CH₃); 2,68 (7a-H) 2.28(7a'-H), 2.55 (7b-H); 2.20 (7b'-H); 1.80 (d, 3H, CH₂ CH₃); -1.70 (s, 2H,2 NH); for the hexyl group, 3.72 (t, 2H, O-CH₂ CH₂); 1.73 (2H, CH₂);1.25 [bs, merged, 6H, (CH₂)₃ ]; 0.78 (t, 3H, CH₃). (see FIG. 1).

2-{1(O-hexyl)ethyl}devinyl pyropheophorbide-a (5): Pyropheophorbide-a(3,200 mg) was reacted with 30% HBr/acetic acid and then with n-hexanolby following the method as discussed for 4 and the desired product wasisolated in 60 to 65% yield. The structure was confirmed by NMRspectroscopy.

EXAMPLE 2 Tumor Treatment

When 2-[1-(O-hexyl)ethyl] devinyl pyropheophorbide-a - structure (5) inScheme 1: S--RO-- where R=(CH₂)₅ CH₃ and m=H, (formula IIa) synthesizedas indicated (5.0 mg) is dissolved in Tween 80 (0.1 ml) and mixed with10 ml Hanks Balanced Salt Solution (HBSS), a solution of approximately0.5 mg/ml in 0.1% Tween 80 is produced after filtration through a 0.22μM Millipore filter. Ten DBA/2 mice with 0.4-0.5 mm diametersubcutaneous SMT-F tumors in the axilla are injected intravenously with0.3 mg/kg body weight of the above solution (after diluting in HBSS sothat the injected volume per mouse is approximately 0.2 ml).Approximately 24 h later the tumor area (having been shaved anddepilated prior to tumor implant) is exposed to laser light at 660-670nm for 30 min at a power of 75 mW/cm² to deliver 135 Joules/cm².Alternately, a Xenon arc lamp filtered to emit a broader band width near670 nm and approximately 283 Joules/cm² can be used.

The day after light treatment all the tumors are seen to be flat(non-palpable) and slight skin blanching over the area is noted. Thisprogresses to frank tumor necrosis over the next few days. At 7 dayspost treatment all tumors remain non-palpable and necrotic. At 30 dayspost treatment, six of the 10 tumors remain non-palpable, and oneremains tumor-free to 90 days post treatment.

EXAMPLE 3 Skin Clearance

Six albino Swiss mice (HaICR) are injected intravenously with a dose of0.1 mg/kg body weight of the compound of formula IIa prepared as inExample 1. After approximately 24 h, the hind foot of the animal isexposed to the same dose of either laser light at 660-670 nm (135Joules/cm²) or the Xenon arc lamp (283 Joules/cm²) as above. Thereaction of the foot is scored for damage over the next few days todetermine the maximum effect, which in this case is a value 0.3equivalent to slight edema. If the internal between the injection andlight treatment is extended to approximately 48 h, the foot reaction iszero (no damage incurred), indicating either clearance or metabolism ofthe sensitizer.

Data obtained as a result of experiment carried out is put forth belowin Table 1.

    __________________________________________________________________________    In Vivo Tumor Photosensitizing Activity of Pyropheophorbide Ethers.sup.1                   Time                        Normal Foot Response.sup.3           Injected Dose                                                                              Interval    Tumor Response.sup.2                                                                          Time Interval                                                                        Maximum                       (mg/kg)      (hours)                                                                            Wavelength                                                                           Day 1                                                                             Day 7                                                                             Day 30                                                                            Day 90                                                                            (hours)                                                                              Reaction                      __________________________________________________________________________    Formula II                                                                    R.sub.5 = --O--(CH.sub.2).sub.5 CH.sub.3                                      R.sub.7 = --CH.sub.2 CH.sub.2 CO.sub.2 H                                      0.05         24   659     0/40                                                                             --  --  --                                       0.1          24   659    6/6 6/6 1/6 1/6 24     0.3                           0.3          24   659    5/5 4/5 0/5 --  48-72  0                             0.3          24   655     0/10                                                                             --  --  --  --     --                            0.3          24   665    10/10                                                                             10/10                                                                              2/10                                                                              0/10                                                                             --     --                            0.3          24   670    10/10                                                                             10/10                                                                              6/10                                                                              1/10                                                                             --     --                            0.3          24   680     8/10                                                                              0/10                                                                             --  --  --     --                            Formula II                                                                    R.sub.5 = --O--(CH.sub.2).sub.5 CH.sub.3                                      R.sub.7 = --CO.sub.2 CH.sub.3                                                 0.3          24   660    6/6 6/6 3/6 3/6 --     --                            0.1          24   660    5/5 3.5 0/5 --  --     --                            Formula II                                                                    R.sub.5 e = --O--CH.sub.3                                                     R.sub.7 = --CO.sub.2 CH.sub.3                                                 0.1          24   660    0/6 --  --  --  --     --                            0.5           3   660    0/6 --  --  --  --     --                            Formula I                                                                     R.sub.1 = --CH.sub.2 O(CH.sub.2).sub.5 CH.sub.3                               R.sub.3 = --CO.sub.2 CH.sub.3                                                 0.3          24   660    6/6 2/6 0/6 --  --     --                            __________________________________________________________________________     .sup.1 SMTF tumor in DBA/2 mice; 135 J/cm.sup.2 light from laser at 75        mW/cm.sup.2                                                                   .sup.2 Number of nonpalpable tumors/Number treated tumors post light          treatment on Day indicated                                                    .sup.3 White Albino Swiss mice; foot exposed using same conditions as for     tumor treatment. Score of 0.3 = light edema; 0 = no reaction.            

The data put forth in Table 1 clearly demonstrates that thepyropheophorbide compounds of the invention are activated by lighthaving a wavelength of about 660 nm. Further, when the compound wereadministered by injection and subjected to light having a wavelength ofabout 660 nm, the treatment was found to be highly effective withrespect to reducing tumor size in as little as seven days.

Further, the data of Table 1 show compounds of the invention clear skinover a period of 24-48 hours after administration. This is a desirablefeature in that the patient is not subjected to prolonged cutaneousphotosensitivity. The data of Table 1 also show that the hexyl ethers offormula II are preferred over methyl ethers in terms of effecting tumorgrowth when used in photodynamic therapy.

While the present invention has been described with reference tospecific compounds, formulations and methods, it is to be understood bythose skilled in the art that various changes may be made andequivalents may be substituted without departing from the true spiritand scope of the invention. In addition, many modifications may be madeto adapt to a particular individual, method of administration, processof synthesizing, etc., which are within the scope of the presentinvention. All such modifications are intended to be within the scope ofthe claims appended hereto.

We claim:
 1. A conjugate which consists essentially of a compound offormula I: ##STR7## wherein --CH₂ R₁ is --CH₂ OR₂ where R₂ is a primaryor secondary alkyl containing 1 to 20 carbons: andR₃ is --CH₂ CH₂ COR₄where R₄ is H or an alkyl group containing from 1 to 20 carbonatoms,covalently bound to a target-specific component.
 2. The conjugateof claim 1 wherein the component is an immunoglobulin or a receptorligand.
 3. A pharmaceutical composition useful for labeling malignanttissue which comprises the conjugate of claim 1 associated with a labelin admixture with a pharmaceutically acceptable excipient.
 4. Aconjugate which consists essentially of a target-specific componentcovalently bound to a compound of formula II: ##STR8## wherein R₅ is--OR₆ where R₆ is a primary or secondary alkyl containing 6 to 20carbons; andR₇ is --CH₂ CH₂ CO₂ R₈ where R₈ is H or an alkyl containing1 to 20 carbons.
 5. The conjugate of claim 4 wherein the component is animmunoglobulin or a receptor ligand.
 6. A pharmaceutical compositionuseful for labeling malignant tissue which comprises the conjugate ofclaim 4 associated with a label in admixture with a pharmaceuticallyacceptable excipient.
 7. A method of treating a human with abnormalcells which replicate at an abnormally high rate, comprising the stepsof:administering to the human a therapeutically effective amount of acompound of formula II ##STR9## wherein R₅ is --OR₆ where R₆ is aprimary or secondary alkyl containing 6 to 20 carbons; and R₇ is --CH₂CH₂ CO₂ R₈ where R₈ is H or --CH₃ ; allowing the compound of formula IIto accumulate on the abnormal cells; and irradiating the compound offormula I with light having a wavelength of about 660 nm, which isabsorbed by the compound of formula I, thereby generating a cytotoxiceffect with respect to the abnormal cells.